Hello Sébastien,

41,547 peak mass shifts are 205 and 197 Daltons, which do not match any common MALDI-TOF contaminant or adduct. Have a look to the following article:

Article Interferences and contaminants encountered in modern mass spectrometry

Furthermore, TFA does not generate any adduct at all in MALDI-TOF MS. On the contrary it may cause ion suppression in ESI MS. Anyway, for MALDI-TOF you may exclude it. Probably you will have the same result without using it for matrix dissolution.

To further investigate what are the peaks in your above spectrum you should check how have you prepared (purification steps) your sample and consider protein isoforms. Maybe you are seeing some post-translation modification.

Good luck.

Please look at the following below links which may help you in your analysis:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3138281/pdf/zbc25451.pdf

Thanks

Hi Sebastien.

You can try different ratios when mixing your sample with sinapinic acid. I used to start with 1:1 or 1:2 (sample:SA) ratio and if the spectra were not great I played a bit with the mix. Some proteins gave better spectra with higher amount of SA. Btw. I agree with Newton, TFA is usually not forming any adducts. There might be some in negative mode but still, it is very rare. And as already mentioned, m/z differences in your spectra does not correspond to TFA.

Dear all,

Thank you for your answers.

This protein is a human protein produced in E.coli. Do you know any PTMs of this size for proteins produced in E.coli? I collect the protein after the production and purification steps done by my colleagues.

I totally agree with you that the shift is +205 and +197 Da, so quite different. However, at these m/z value, the resolving power is quite bad on a MALDI-TOF, which means that the peak separation and m/z value can be easily wrong... Also the top of the two peaks is not easily determine.

Best,

Sébastien

Sébastien, below is a list of mass changes ascribed as PTMs:

https://www.sigmaaldrich.com/life-science/proteomics/post-translational-analysis/phosphorylation/mass-changes.html

As Lucas Krasny mentioned above you may change sample:matrix ratios and see if your signal resolution improves. You may also try other matrices, as DHAP or THAP - if your protein is glycosilated you may get a better signal resolution using some of those matrices.

In contrast, it is correct that TOF resolution in linear mode is quite limited at such a mass range - there is not much to do about that. If you want to seriously improve your peak resolution you will have to use another analyzer, such as FTICR or Orbitrap.

Hi Sébastien Brûlé

PTMs in E-coli are minimum, did you repeat this analysis, do you see these additional peaks reproducibly.

Have a look at this site for probable PTMs https://abrf.org/delta-mass

Dear Sébastien,

Despite the protein you analyzed is produced by bacteria, it looks very like that you have an glycoprotein. MW of the common monosaccharide residue, N-acetylglucosamine (or N-acetylhexosamine) is about 203 Da that has a good correlation with your data. Try to treat you sample with exoglycosidase that cuts this residues (for example, β-N-Acetylglucosaminidase) and repeat the analysis.

But in a more classical way, firstly you need to do is to measure MW with a better mass accuracy and resolution (ESI-TOF or orbi) and secondly perform a bottom-up approach with several proteases to reliable identify this modification.

Best wishes,

Maksim Degterev

My mass-spec knowledge is rather rudimentary.

But besides cellular PTM, did you look at possible production-process related modifications? Oxidation, amidation, or a combination thereof.

If urea was used during the purification, could carbamylation be in the mix?

Dear Achim Recktenwald,

It is unlikely that the recombinant protein will be full of the variuous modifications ans still have a good MALDI spectra with resolved peaks of modified forms. Highly probable that in this case one peak is equal to one modofication.

Best,

Maksim Degterev

Hello all,

Here is a paper about the presence of covalent adduct produced when the sinapinic acid matrix is used. the m/z of the covalent adduct is 208.

https://link.springer.com/article/10.1007%2Fs13361-012-0490-z

I do not know if I am in presence of that but I keep it in mind. Also, do you work with other matrix than SA for big proteins (at least 40 kdA)?

Best,

Sébastien

At such mass range with linear mode TOF analyser you don't have enough resolution to conclude what your additional peaks are, so 205 and 208 Dalton peaks can't be separated on your experiment in any condition. You should have a look to FWHM resolution definition.

For other matrices please see my answer above.

Hi all,

I get the same pattern for other proteins. So I decided to change my sinapinic acid preparation.

I spotted 1 µL of sample + 1µL 50% ACN 0.1% TFA in water (original matrix) and another position with 1 µL protein + 1 µL 50% ACN 0.15 % Formic acid.

There is no difference. The problem does not come from the acidic solution... I am still investigated if it is due to my chemicals compounds.

Sébastien

Please find attached another example from a protein produced in E.coli by a colleague.

any suggestion?

Sébastien

You are seeing the isotopic peaks of your protein. It is quite often with good preparations in Ultraflex mass spectrometers with linear mode analysis.

You should have a look to laser power/offset in your instrument. Laser power should be set to the minimum requirement.

Hi, I do not think that this is isotopic peaks at this high molecular mass.

However, I found a publication that shows the possible presence of sinapinic acid adducts with a +206 m/z shift.

Article Possible Evidence of Amide Bond Formation Between Sinapinic ...

https://www.cigs.unimo.it/CigsDownloads/labs/malditof/didattica/MALDI_MS_Tutorial.pdf (page 49)

Best,

Sébastien

Hi Sebastien, if so (sinapinic adducts) you should not see any additional peaks if using DHB or DHAP matrices, for instance.

You are right!

I have just tested on the standard 2 of bruker.

Please find attached a quick measurement.

In red 1500 shots with HCCA, in blue 1500 shots with sinapinic acid.

The result is clear, there are less adducts in presence of HCCA.